Methods to identify agents that bind the flap-tip helix of RNA polymerase

ABSTRACT

The invention provides methods to identify moieties which specifically bind the flap-tip helix of the β subunit of RNA polymerase and to identify inhibitors of the interaction between those moieties and the flap-tip helix.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.application Ser. No. 60/286,783 filed on Apr. 25, 2001, under 35 U.S.C.§ 119(e), the disclosure of which is incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made at least in part with a grant from theGovernment of the United States (grant GM 38660 from the NationalInstitutes of Health). The Government has certain rights to theinvention.

BACKGROUND OF THE INVENTION

DNA, RNA, and regulatory molecules control gene expression throughinteractions with RNA polymerase (RNAP). RNA polymerase is anevolutionarily conserved, multisubunit enzyme responsible for utilizinga DNA strand as a template and making a complementary RNA molecule inall known organisms. As such, it is central to all gene transcriptionand must function for the cell to survive.

The transcription cycle can be divided into major phases: promoterengagement, initiation, RNA chain elongation, and termination. Promoterengagement encompasses several steps: promoter location and recognitionby the RNA polymerase holoenzyme (core enzyme complexed with one ofseveral σ factors), initial and reversible binding of RNA polymerase toduplex promoter DNA (closed complex formation), and formation of an opencomplex in which about 12 bp of DNA, including the transcription startsite, are melted. At least four important intrinsic inputs affectpromoter engagement: the hexamer centered at position −10 upstream fromthe transcription start site (−10 region), the hexamer at position −35(−35 region), the region of DNA between these two elements (spacerregion), and a region located between −40 and −60 (the UP element).

The rates at which these steps occur are dictated by both extrinsic andintrinsic interactions. Extrinsic inputs include protein-proteincontacts made by activators and repressors that bind in the vicinity ofthe promoter DNA and can modify the rates of either closed- oropen-complex formation or promoter escape. Intrinsic contacts are madeby the σ subunit of RNA polymerase to the −10 and −35 regions of the DNAand sometimes by the α-subunit C-terminal domain to the UP element (Rosset al., 1993).

After the open complex has bound the initiating NTPs, it becomes aninitial transcription complex and can follow several alternativereaction pathways: (i) the synthesis and release of short (2 to 8nucleotides [nt]) RNA transcripts (abortive initiation); (ii)reiterative synthesis resulting in homopolymer extensions of the initialRNA transcripts (stuttering); and (iii) release of σ, translocation awayfrom the promoter, and formation of a transcription elongation complex(TEC) with loss of upstream DNA contacts, usually when the transcript is8 to 9 nt in length (promoter escape).

Once RNA polymerase converts from initial transcription complex to TEC(i.e., escapes the promoter), it becomes stably associated with the RNAand DNA chains and can elongate the RNA chain 30 to 100 nt/sec in vivo.Two distinct types of translocations in the TEC must occur to allow thisrapid movement; (i) translocation of the RNA 3′ end from position i+1 toi in the active site (the 3′-terminal nucleotide is the index position)as successive nucleotides are added and (ii) translocation of DNA andRNA chains through RNA polymerase (RNA and DNA translocation).

RNA chain elongation is punctuated by certain sites where nucleotideaddition is slowed by pause, arrest, and termination signals. Pausesignals cause. RNA polymerase to isomerize from the rapidly elongatingTEC to alternative conformations in which RNA chain extension isreversibly inhibited (by factors of 10² to 10⁴). Termination signalscause the release of RNA and DNA and can be positively or negativelyregulated by a variety of extrinsic inputs.

When RNA polymerase encounters a termination signal, RNA polymerasestops adding nucleotides to the RNA, separates the DNA-RNA hybrid,releases the newly synthesized transcript, and dissociates from the DNAtemplate. Three types of termination signals for bacterial RNApolymerase have been described: (i) intrinsic terminators (ρ-independentterminators) which require a stable RNA hairpin formed 7 to 9 nt fromthe terminated RNA 3′ end and immediately followed by at least 3 Uresidues, but no extrinsic factors (reviewed in Platt, 1997; Richardsonet al., 1996; Roberts, 1996; Uptain et al., 1997); (ii) ρ-dependentterminators, which depend on the presence of p factor, a hexamericRNA-binding protein with ATPase activity (reviewed in Platt, 1997;Richardson et al., 1996); and (iii) persistent RNA-DNA hybridterminators at which pairing of nascent RNA to the template justupstream from the TEC dissociates a complex containing 3′-proximalU-rich RNA (Tomizawa et al., 1997).

E. coli NusA is a 55-kDa acidic protein that interacts with ρ, λ N, andRNA through one or more interaction regions and RNA polymerase throughcontacts to the α-subunit C-terminal domain and either β′ or β (Liu etal., 1996; Richardson et al., 1996). NusA enhances both pausing andp-independent termination in the absence of other cellular or phageproteins, is found in all prokaryotes and archaebacteria sequenced todate, and is essential in E. coli unless ρ activity is reduced by amutation (Zheng et al., 1994).

A universal feature of RNA polymerases is a regulated conversion from aninitiating form that holds the RNA weakly, to an elongating form wherethe enzyme holds RNA tightly during RNA synthesis, and then back to aterminating form that releases RNA. The molecular basis of the switchfrom initiation to elongation to termination is unknown. However,conservation from bacteria to humans of RNA polymerase's core subunitcomposition (β,βα₂ in bacteria), amino-acid sequences, three-dimensionalstructure, and contacts to DNA and RNA suggest that the switch will besimilar for all multisubunit RNA polymerases (Zhang et al., 1999; Crameret al., 2000; Korzheva et al., 2000).

A nascent RNA hairpin can terminate transcription by bacterial RNApolymerase if the hairpin includes the 3-5 nt usually found in the RNAexit channel and disrupts at least one base pair (bp) of the about 8-bpnascent RNA:template DNA hybrid that is present in stable TECs (Nudleret al., 1997; Sidorenkov et al., 1998; Artsimovitch and Landick, 1998;Yarnell and Robert, 1999). Similar RNA hairpins can also pause, ratherthan terminate, transcription when they form more upstream but near theRNA:DNA hybrid, rather than invade it. Both hairpin-dependent pausingand termination can be enhanced by the universal bacterial protein NusA(Chan and Landick, 1993; Sigmund and Morgan, 1988).

Two models can explain hairpin effects on transcription (Korzheva, 2000;Yarnell and Roberts, 1999; Farnham and Platt, 1980; Yager and vonHippel, 1991; Gusarov and Nudler, 1999; Mooney and Landick, 1999;Davenport et al., 2000; Artsimovitch and Landick, 2000). In therigid-body model, a pause or terminator hairpin begins forming when onlyits loop and upper stem have emerged from the exit channel and thenpulls RNA through the channel and away from the active site to avoidsteric clash with a rigid RNA polymerase as the lower stem pairs. Thispartially unwinds the RNA:DNA hybrid and moves RNA polymerase forwardwithout nucleotide addition (the hybrid is wedged against the upstreamedge of the active-site cleft in a TEC; see FIG. 4A in Korzheva et al.,2000). In the allosteric model, once the hairpin starts to form itinstead triggers a conformational change in RNA polymerase that inhibitsnucleotide addition in the active site and reduces affinity for productRNA, without necessarily moving the intervening RNA. However, it isunknown which model explains hairpin effects on transcription.

As transcription is central to gene regulation, a better understandingof the transcription process and the cellular factors which interactduring transcription could lead to the identification of specificinhibitors of transcription. Thus, what is needed is a method todetermine what factors specifically interact with portions of RNApolymerase during transcription. What is also needed is a method toidentify agents that specifically inhibit those interactions.

SUMMARY OF THE INVENTION

The invention provides a method to identify agents that specificallyinhibit the interaction of moieties, including but not limited toprokaryotic cellular proteins, e.g., transcription factors or subunitsof RNA polymerase such as the β′ subunit, nucleic acid aptamers andpeptide aptamers, with the flap-tip helix of the β subunit of core RNApolymerase. The flap domain of the β subunit was identified from thecrystallized structure of Thermus aquaticus (Zhang et al., 1999) RNApolymerase. At the upper most tip of this flap is a domain termed theflap-tip helix because of its helical structure. As describedhereinbelow, a short α-helix at the tip of the flap-like domain thatcovers the RNA exit channel of RNA polymerase contacts a nascent RNAstem-loop structure (hairpin) that inhibits transcription, and thisflap-tip helix is required for activity of the regulatory protein NusA.In particular, a short nine amino acid segment deletion or alternativelymutation of four hydrophobic amino acids within the flap-tip helix,prevents the action of the a initiation factors and the elongationfactor NusA. It is highly likely that the flap-tip helix contacts themain body of RNA polymerase during RNA chain elongation, thussequestering the exiting nascent RNA chain under the closed flap domain.During initiation, the flap-tip helix instead appears to contact σ toopen the flap domain. At pause and termination sites, NusA may helpreopen the flap domain through a contact to the flap-tip helix.Protein-RNA crosslinking, molecular modeling, and effects of alterationsin RNA polymerase and RNA all suggest that a tripartite interaction ofRNA polymerase, NusA, and the hairpin inhibits nucleotide addition inthe active site, which is located 65 Å away. These findings favor anallosteric model for regulation of transcript elongation.

As also described hereinbelow, expression of β subunits containingflap-tip helix mutants is lethal to bacterial growth. The lethalamino-acid substitutions occurred at four hydrophobic residues on oneface of the flap-tip helix (L901, L902, I905, and F906 of the β subunitof E. coli RNA polymerase; FIG. 5B). These amino acid residues arehighly conserved in bacteria, but not in eukaryotes, and together form ahydrophobic patch that makes alternate contacts to σ factors, NusA, andthe main body of RNA polymerase. As RNA polymerase is a largemulti-subunit complex (having about 3300 amino acids), theidentification of the region of the core RNA polymerase whichspecifically interacts with transcription factors represents asignificant finding as it provides a specific target for drug discovery.

Thus, the properties of the flap-tip helix make it an ideal candidatefor rational design of a drug, e.g., an antibiotic, as any agent thatspecifically binds to the flap-tip helix should block function oftranscription factors essential for bacterial viability and thus thoseagents will inhibit a wide variety of bacterial RNA polymerases and killa wide variety of bacteria. Further, because the flap-tip helixinteracts with multiple essential transcription factors, it should berelatively difficult for bacteria to acquire natural resistance toantibiotics targeted to the flap-tip helix by mutations that alter theamino-acid sequence of the flap-tip helix. Such mutations would alsocompromise interactions with the essential transcription factors andthus block their function. Hence, the flap-tip helix is a novel targetfor design of new antibiotics that is highly likely to yield drugs thatwill be effective against a wide variety of bacterially caused diseases.

Thus, the invention provides a method to identify one or more agentswhich inhibit or prevent the binding of a moiety to the flap-tip helixof the β subunit of core RNA polymerase. In one embodiment, the methodcomprises contacting the one or more agents with isolated core RNApolymerase, or an isolated β subunit of RNA polymerase or a portionthereof which comprises the flap-tip helix, so as to form a complex. Asused herein, “isolated and/or purified” refers to in vitro preparation,isolation and/or purification of a peptide, protein or a complex ofbiomolecules, e.g., core RNA polymerase, so that it is not associatedwith in vivo substances or is substantially purified from in vitrosubstances. The portion of the β subunit may include the flap domain,e.g., residues corresponding to residues 830 to 1085, or flap-tip helix,e.g., residues 900 to 907, residues 897 to 907, residues 900 to 909, orresidues 886 to 953 of β of E. coli RNA polymerase. Preferably, theportion of the β subunit which includes the flap-tip helix comprises atleast 8, more preferably at least 10, and even more preferably at least20, residues, although smaller fragments are also envisioned. Thecomplex is contacted with a moiety, e.g., an isolated moiety, whichspecifically binds the flap-tip helix and then it is detected ordetermined whether the one or more agents inhibit or prevent the bindingof the moiety to the flap-tip helix. In another embodiment, the one ormore agents is contacted with a moiety, e.g., an isolated moiety, whichspecifically binds the flap-tip helix so as to form a complex. Forexample, the moiety may be a transcription factor such as an initiationfactor, e.g., σ, an elongation factor, e.g., NusA or NusG, or ananti-termination protein including λ N or Q or rfah proteins, or anothersubunit of RNA polymerase such as the β′ subunit. For example, the β′subunit-flap-tip helix interaction may be important for stabletranscription elongation complexes. The complex is contacted withisolated core RNA polymerase, or an isolated β subunit of RNA polymeraseor a portion thereof which comprises the flap-tip helix. Then it isdetected or determined whether the one or more agents inhibit or preventthe binding of the moiety to the flap-tip helix.

The detection or determination of binding or inhibition or preventionthereof can be accomplished by a variety of methods some of which aredescribed herein. For example, core RNA polymerase, the β subunit or aportion thereof may be labeled or may bind to a detectable label such asa labeled antibody. Alternatively, or in addition to, the moiety may belabeled or bind to a detectable label. Thus, assays such as fluorescenceresonance energy transfer assays, luminescence resonance energy transferassays, cleavage assays (protease or nuclease cleavage), crosslinkingassays, scintillation proximity assays or fluorescence perturbationassays and the like may be employed.

In another embodiment, the agents may be identified in vivo. Forexample, the invention also provides a method in which one or moreagents is contacted with a recombinant cell. The recombinant cellexpresses a first fusion polypeptide and a second fusion polypeptide.The first fusion polypeptide comprises the flap-tip helix and a firstpolypeptide and the second fusion polypeptide comprises the prokaryoticprotein or a fragment thereof which specifically binds the flap-tiphelix and a second polypeptide which is a ligand for the firstpolypeptide. The binding of the first polypeptide and the secondpolypeptide yields a detectable signal. Then it is detected ordetermined whether the one or more agents inhibits or prevents thesignal. An exemplary method employs a first or the second polypeptidewhich comprises a DNA binding domain while the other polypeptidecomprises an activation domain.

Also provided is one or more agents identified by the methods of theinvention. Further provided is a method of using those agents. Themethod comprises contacting a cell with the agent and detecting ordetermining whether the agent inhibits or prevents the growth of thecell. Preferably, the agent inhibits or prevents the growth of aprokaryotic cell but not a eukaryotic cell.

The invention also provides a method to identify moieties whichspecifically bind the flap-tip helix. In one embodiment, a peptidecomprising the flap-tip helix, e.g., residues corresponding to residues886 to 953 of β of E. coli RNA polymerase, or a portion thereof iscontacted with the one or more moieties. Then it is determined whetherthe one or more moieties specifically bind to the flap-tip helix. In oneembodiment, the moiety is present in the surface of a recombinant phage,virus, or cell. Thus, a library of molecules expressed by recombinantphage, virus or cells, e.g., bacteria or yeast, is contacted with apeptide comprising the flap-tip helix or a portion thereof and moleculesthat specifically bind to the peptide identified and optionallyisolated.

The invention thus further provides an isolated and purified portion ofthe β subunit of RNA polymerase which binds to the β′ subunit, NusAand/or σ. Preferably, the portion comprises residues corresponding to886 to 953, residues 897 to 907, or residues 900 to 909 of the β subunitof E. coli RNA polymerase.

The invention further provides agents identified by the methods of theinvention and, in particular, agents which inhibit the growth ofprokaryotic cells which are associated with disease, see e.g., ZinsserMicrobiology (17th ed., Appleton-Century-Crofts, N.Y. (1980).

Also provided is a method to identify one or more agents which inhibitor prevent transcription from a promoter that is dependent on a −35sequence for activity. The method comprises contacting the one or moreagents with a composition for transcription comprising wild-type RNApolymerase and a first construct comprising a first promoter that isdependent on a −35 sequence to promote transcription and operably linkedto an open reading frame for a gene. Then an agent is identified thatinhibits or prevents transcription from the first construct relative totranscription by wild-type RNA polymerase from a second constructcomprising a second promoter that promotes transcription independent ofthe presence of a −35 sequence, which second promoter is operably linkedto an open reading frame for a gene, thereby identifying an agent thatinhibits or prevents transcription from a promoter that is dependent ona −35 sequence for activity. In one embodiment, the one or more agentsare contacted with a recombinant cell augmented with the firstconstruct. Preferably, the level of transcription from the firstconstruct in the presence of the agent is substantially the same as thelevel of transcription by a mutant RNA polymerase comprising a mutantflap-tip helix from a third construct comprising a promoter that isdependent on a −35 sequence to promote transcription and operably linkedto an open reading frame for a gene. Also preferably, the agent does notinhibit transcription by a mutant RNA polymerase comprising a mutantflap-tip helix from a third construct comprising a promoter thatpromotes transcription independent of the presence of a −35 sequence andoperably linked to an open reading frame for a gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Pause hairpin crosslinking. (A) Structures of wild-type (SEQ IDNO:3) and tetraloop (SEQ ID NO:4) his pause RNAs with positions ofanalog (magenta) or ³²P (*) incorporation, the −11 and +1 nt (green),the spacer RNA present in the exit channel (color-on-black), and theRNA:DNA hybrid (underlined) indicated. (B) Amino acid sequence (SEQ IDNO:5) around the previously identified crosslink target in E. coli RNApolymerase (β903-952; Wang et al., 1997). —Δ—, flap-tip deletion. Likelytargets of 5-iodoU crosslinking are in red. (C) Crosslinking of thetetraloop pause hairpin to wild-type, βF906A and βF934A RNA polymerases.TECs containing 5-iodoU and [³²P]CMP (FIG. 1A) were halted at −9, −3,and the pause detected as described by Wang et al. (1997). Afterirradiation at 308 mm, samples were denatured, subjected to SDS-PAGE,and visualized by phosphorimaging.

FIG. 2. Model of pause hairpin formation in a TEC. (A) RNA polymerase(α; white; β, light blue; β′, pink) is rendered semitransparent toreveal key internal features and contents of the active-site cleft. Theflap domain (dark blue worm) and clamp (violet) domains fill the RNAexit channel. The flap domain connects to K1065 and E813/D814 (bluespacefill) in the active site (Mg²⁺, magenta; NTP, green) through theantiparallel beta-sheet connector (green). Flap-tip residues deleted inβΔ(900-909) are in green. Template DNA (orange), nontemplate DNA(yellow) and RNA (red) are positioned as in Korzheva et al. (2000); thepause hairpin is magenta. The dotted arrow indicates likely closure ofthe exit channel. (B) Pause hairpin-RNA polymerase interaction magnifiedand rotated left 30° and down 60° relative to A. Upstream DNA has beenremoved for clarity. The tetraloop pause RNA hairpin was positioned bythe −11 nt (green) and the loop uridines (yellow) that crosslink to F906(green spacefill). Hydrophobic-patch residues (L901, L902, I905; green)may contact β or β′ (dotted line) in a TEC. Possible NusA interactionsare indicated with arrows.

FIG. 3. The −11 nt does not move upon paused TEC formation. (A)Phosphorimage of β and β separated by SDS-PAGE after crosslinking inpaused and nonpaused (with antisense oligonucleotide) TECs with 4-thioUat −11 or 5-iodoU at the 3′ end. (B) Single-hit, partial CNBr cleavageof β′ and β subunits crosslinked by 5-iodoU at −11 in paused andnonpaused TECs (Korzheva et al., 2000). Crosslinks were assigned betweenthe cleavage site closest to the N- or C-terminus that removed theradioactive label and the adjacent site at which cleavage yielded aradioactive fragment. (C) Location of the −11 nt (green) in paused andnonpaused TECs based on results in B. Orange, region of β′ crosslink.Blue, region of β crosslink. Magenta, the 6 Å by×9 Å area in which themapped regions of crosslinking to β and β′ overlap. The flap is removed(dark green outline) to reveal an extended, single-stranded RNA (red) inthe exit channel.

FIG. 4. Effects of hairpin-spacing, stem-length, flap-tip helix, andNusA on pausing. (A) Pause half-lives (gray bars) were measured asdescribed previously (Yarnell and Roberts, 1999). The residual half-lifewith antisense oligonucleotide (blue) or 1 M KCl (red) reveals thehairpin's contribution to pausing (Artsimovitch and Landick, 2000).Antisense oligonucleotides for the tetraloop (SEQ ID NO:6) and 8-bp (SEQID NO:7) stem pause sites were extended to include pairing to thehairpin loop. (B) TECs with 5-iodoU and ³²P in the hairpin loop (FIG.1A) were halted at −9 or the pause, combined with antisenseoligonucleotide or NusA as indicated, irradiated at 308 nm, subjected toSDS-PAGE, and phosphorimaged.

FIG. 5. (A) Location of the flap-tip helix (green) in the transcriptionelongation complex. RNA polymerase (β′ subunit, pink; β subunit, blue,at and omega subunits, gray) binds to about 30 bp of DNA (template DNAstrand, orange; nontemplate DNA strand, yellow) and melts about 17 bp toform a transcription bubble, within which 8 nt of template DNA pair tothe 3′-proximal 8 nt of RNA transcript (red) to form an RNA:DNA hybrid.The RNA exits underneath the flap domain, which is thought to close overthe exiting RNA by a contact of the hydrophobic patch on flap-tip helixto β′ or β. (B) Magnified view of the flap-tip helix and possible rolesin RNA polymerase function. Orientation is same as shown in A. Both σinitiation factors and the NusA elongation factor require the flap-tiphelix for function, most likely because σ factors open the flap domainthrough contact to the flap-tip helix for initiation and because NusAmay stabilize partial reopening of the flap-domain at pause andtermination sites during RNA chain elongation. Substitutions of L901,L902, I905 and F906 are all lethal for bacterial growth, establishingthe essential nature of the hydrophobic patch on the flap-tip helix. Inthe absence of σ or NusA, the hydrophobic patch likely contacts β or β′at the positions shown.

FIG. 6. Mapping of the pause hairpin loop crosslink to β904-908. (A)Trypsin cleavage of 5-iodoU-crosslinked β in K909C RNA polymerase andNTCB cleavage of 5-iodoU-crosslinked β in wild-type and K909C RNApolymerase. (B) Trypsin cleavage of 4-thioU-crosslinked β in K909C RNApolymerase and NTCB cleavage of 4-thioU-crosslinked β in wild-type andK909C RNA polymerase. (C) Segments that retain radioactive label(magenta) after digestion of β with trypsin or partial digestion withNTCB. The overlap among these segments restricts the possible locationsof the 5-iodoUi and 4-thioU crosslinks to β904-908. (D) Comparison ofcrosslinking by 5-iodoU and 4-thioU to RNA polymerase in wild-type andF906A paused TECs.

FIG. 7. Mapping of the −11 nt crosslink in paused and nonpaused TECs.(A) Partial CNBr cleavage of β¢ after crosslinking with 4-thioU placedat the −11 nt in paused and nonpaused TECs (without and with antisenseoligonucleotide, respectively). M, control CNBr cleavage of N-terminallylabeled β′ subunit. (B) Partial CNBr cleavage of β after crosslinkingwith 4-thioU placed at the −11 nt of paused and nonpaused TECs. M,control CNBr cleavage of C-terminally labeled β subunit. (C) NormalizedCNBr cleavage of −11-nt-crosslinked β′ and β, respectively. Cleavage of³²P-end-labeled β′ or β was used to normalize cleavage of subunitscrosslinked in paused (black) and nonpaused (with oligonucleotide,white) TECs. Significant drops in the normalized intensity (e.g.,between β¢298-237 and β1243-1273, colored bars) reveal the crosslinkpositions (Korzheva et al., 2000).

FIG. 8. Effect of NusA on pausing and transcription by β D(900-909) RNApolymerase. (A) Procedure for assembly of TECs and assay of pausing. (B)Denaturing polyacrylamide gel of RNAs synthesized by wild-type orβD(900-909) mutant RNA polymerases on the synthetic templates with orwithout NusA. When present, NusA was added to 90 nM immediately beforeelongation of C24 TECs. Samples were removed at 5, 10, 20, 40, 90, 180,and 360 seconds after addition of all 4 NTPs, denatured at 95° C. for 2minutes after addition of urea to 4 M, and separated in a 10%polyacrylamide gel containing 8 M urea. Pause half-lives were calculatedas described previously (Landick et al., 1996).

FIG. 9. Summary of the transcription cycle. RNA polymerase (RNAP) bindsto σ to form holoenzyme, which then recognizes and binds to promotersequences. After formation of an initial RNA transcript up to about 10nt in length, RNAP releases σ and forms a TEC. NusA interacts with theTEC to regulate RNA chain elongation. Upon termination, RNA, DNA, andNusA are released from RNAP and the core enzyme, consisting of two αsubunits and one each of the β, β′, and ω subunits is free to begin thecycle again (ω is dispensable in E. coli.) FIG. 10. Location andsequence of the flap-tip helix. The flap domain forms a part of RNAP atthe end of the active-site channel (transcription occurs from left toright). σ factors contact the clamp and flap domains. The flap-tip helixis generally located between P897 and G907. The hydrophobic patch (SEQID NO:8) includes residues L901, L902, I905, and F906 which lie on theside of the flap-tip helix that faces away from RNAP.

FIG. 11. Plasmids that express RNAP subunits. pRL702 expresses only theβ subunit from the rpoB gene under the control of the trc promoter, andthe β subunit carries the hexahistidine tag at its N-terminus. pIA312expresses all the core RNAP subunits (α, β, and β′) from the T7 RNAPpromoter, allowing assembly of an E. coli RNAP bearing an intein and achitin-binding domain (CBD) at the C-terminus of the β′ subunit in cellsthat express T7 RNAP. The intein and CBD allow efficient purification ofRNAP and are cleaved off by DTT treatment β produced from pIA312 doesnot carry a hexahistidine tag, but all pIA312 derivatives prepared bytransferring the NcoI to Sse83871 fragment from pRL702 to pIA312 carrythe hexahistidine tag on β.

FIG. 12. Effect of flap-tip substitutions and a deletion on recognitionof promoters that do (T7 A1) or do not (Gal P1 consensus −10) depend ona −35 promoter element. A) and C). Relative levels of transcriptionproducts. B) and D). Sequences of the 17 A1 and GalP1 consensus −10promoter templates (SEQ ID NOs:9 and 10, respectively). Downstream DNAcontinues another approximately 300 bp past the sequences shown,however, the transcripts stop at +29. The RNAs synthesized on thesetemplates are shown in italics above the DNA sequences.

FIG. 13. Trypsin cleavage of the RNAP β subunit in the absence andpresence of σ⁷⁰. The plots below the gel images show the rates of βsubunit and σ⁷⁰ cleavage. Protection of the β subunit flap-tip (SEQ IDNO:8) and σ⁷⁰ against trypsin cleavage (panels A & B) is lost when theLQ901 substitution is present in the β subunit (panels C & D).

FIG. 14. Trypsin cleavage of the flap-tip helix in the absence andpresence of NusA.

DETAILED DESCRIPTION OF THE INVENTION

Bacterial RNA polymerase synthesizes RNA from cellular genes and thusplays a central role in the regulation of gene expression. Core RNApolymerase can synthesize RNA but is unable to specifically bind DNA atpromoter sites. To specifically bind DNA and initiate transcription ofgenes, a binds to core to form the holoenzyme. Each step in initiation,elongation and termination are potential sites for inhibitors oftranscription.

Gene expression often is regulated during RNA chain elongation.Regulation depends both on interruptions to transcription caused bypause, arrest, and termination signals encoded in the DNA and RNA and onauxiliary proteins that modify the response of RNA polymerase (RNAP) tothese signals. Pausing (a temporary delay in chain elongation)synchronizes transcription and translation in prokaryotes, slows RNAP toallow timely interaction of regulatory factors, and is a precursor toboth arrest (complete halting without dissociation; Komissarova andKashler, 1997; Mote and Reines, 1998), and dissociation of thetranscription elongation complex (TEC) at ρ-dependent and ρ-independentterminators (Richardson and Greenblatt, 1996).

Numerous auxiliary proteins modulate pausing in organisms from bacteriato humans. Two of these, NusA and NusG, are universally conserved amongbacteria and archaebacteria (Ingham et al., 1998) are typicallyessential to cell viability, and, respectively, inhibit or stimulatepausing by bacterial RNAP (Burns et al., 1999). NusA and NusG alsomodulate the termination activity of ρ protein (which dissociates pausedTECs) and, together with other auxiliary proteins like λ N or Q,assemble antitermination TECs that resist pausing and termination.

Associated with a σ factor, bacterial core RNAP first binds to 40-60 bp,of duplex promoter DNA. In a series of at least three transitions thatincludes downstream extension of its footprint on DNA and extensiveburial of nonpolar surface with significant protein conformationalchanges, σ-RNAP separates the double helix, placing the template DNAstrand into the active site channel (Craig et al., 1998 and referencestherein). These transitions are orchestrated via specific interactionsof σ with the melted nontemplate strand and upstream duplex DNA, and theα subunit C-terminal domain (not visible in the structure) with DNAfurther upstream. Thus, contacts to DNA in the promoter and initiatingcomplexes span 70-90 bp (235-305 Å versus RNAP's maximal dimension of150 Å), requiring that DNA wrap around RNAP (Craig et al., 1998 andreferences therein). The initiating complex can synthesize and releaseRNAs up to about 10 nt in length apparently without loss of theseextensive contact during abortive initiation.

Further transcription requires rearrangement to a highly stable TEC. TheTEC must successfully traverse over 10⁴ (in bacteria) to 10⁶ (ineukaryotes) bp without dissociation (RNAPs cannot resume synthesis ofRNAs once they are released). In bacteria, this transition isaccomplished by breaking contacts of σ to the promoter and to core RNAP,threading RNA into an exit tunnel, and rearranging DNA contacts so thatthe footprint is reduced to only about 35 bp (−17 to +18). Formation ofa eukaryotic TEC additionally required multiple phosphorylation of aheptapeptide repeat at the C terminus of its largest subunit by specifickinases. This phosphorylation is key to an activating transition inwhich association of special factors equip RNAPII for long-distancetranscription on a chromatin template (see Uptain et al., 1997;Shilatifard, 1998 and references therein).

To terminate transcription, the stability of the TEC must decrease. Inbacteria, this can be accomplished by an intrinsic termination signalconsisting of a G+C-rich RNA hairpin followed by 7-9 nt of U-rich RNA.In eukaryotes, the mechanism of termination is unknown, but appears torequire loss of some factors and possibly the action of others thatdissociate the complex (see Uptain et al., 1997).

The new Taq RNAP and yRNAPII structures confirm the basic RNAParchitecture inferred from the earlier EM results and reveal importantfeatures not previously resolved. Both core RNAPs contain the “jaws”previously seen open or closed in different structures (plus and minus afor E. coli RNAP; in different crystal forms of core yRNAPII). Theincreased resolution of the new structures reveals that the jawsactually consist of an upper hinged arm and a broader underlying shelf.In the Taq RNAP structure, this hinged arm (β domain 2) appears open,even though σ appears absent. In both yRNAPII structures the arm isclosed.

Zhang et al. (1999) definitively located the active site in a deeppocket at the junction of the jaws, the same location inferred from theyRNAPII TEC structure by Poglitsch et al. (1999). What was previouslythought to be a continuous 25 Å channel extending from the jaws throughthe main body of the enzyme (through which duplex DNA or RNA-DNA duplexcould pass) is actually blocked within the jaws of both RNAPs bysignificant protein density (comprised of β′ regions F and G in TaqRNAP, Zhang et al., 1999; see also FIG. 5A in Poglitsch et al., 1999 andFIG. 6 in Fu et al., 1999). Moreover, the channel consists of two parts,a 10-14 Å “secondary” channel formed by the β′FG “wall” and the insideof the jaws, and a main channel extending away from the active site inthe upstream direction (the active site channel). Both groups proposethat the “secondary” channel could serve as an entry site for NTPsubstrates. As shown in the yRNAPII TEC structure, downstream DNA entersRNAP parallel to and between the jaws, rather than encircled by the jaws(Poglitsch et al., 1999). The upstream DNA appears to exit RNAP at anapproximate 90° angle relative to the path of the downstream DNA.

Both groups also propose a similar path for the nascent RNA-template DNAhybrid within the active site channel, which in Taq RNAP routes the RNAthrough a pocket in β that binds rifampicin (an inhibitor that blocksinitiation by bacterial RNAP). Separation of the RNA:DNA hybrid wouldoccur near a feature called the “rudder” in Taq RNAP, which is notresolved or not present in the yRNAPII structure. After separation, theRNA transcript is thought to exit RNAP in a tunnel; both the bacterialand eukaryotic structures contain a putative RNA exit tunnel composed ofa groove covered by a mobile domain. In Taq RNAP, the flap-like domain(β domain 6) connects to the body of the enzyme via a hinge-like stemand could close over the exiting RNA from above the tunnel. In yRNAPII,an apparently larger hinged domain reaches over the RNA exit tunnel frombelow. The yRNAPII hinged domain is significantly displaced away fromthe exit tunnel by crystal packing forces in the X-ray structure(relative to its position in the TEC EM structure), confirming that thisdomain is mobile (see FIG. 5 in Fu et al., 1999). The apparentdifferences in the RNA exit tunnels of bacterial and eukaryotic RNAPsmay have functional significance.

Previous biochemical studies indicate that or may contact the coreenzyme in three locations whose movement relative to one another couldopen and close RNAP's active site channel, which encloses the RNA:DNAhybrid, and RNA exit tunnel, which encloses the exiting RNA. On contactis to the very tip of the flap domain (Fisher and Blumenthal, 1980;Borukhov et al., 1991). As described above and by Zhang et al. (1999),the flap (β domain 6) might open and close over the exiting RNA. Theother two contacts are to parts of RNAP that could close the active sitechannel: the prominent coiled-coil in β′ that projects from the rudderon the lower surface of the active site channel (Arthur and Burgess,1998), and two helices (β domain 3) that cap the active site channel inthe Taq RNAP structure (Owens et al., 1998). If RNAP assumed a closedconformation, these two pairs of α helices could approach each other atthe upstream edge of the active site channel, conceivably stabilizingthe position of the rudder and locking the channel in the closedconformation.

It is striking that the active site channel in the Taq RNAP structure ismore open than needed to fit an 8 bp RNA:DNA hybrid. The ability ofmajor domains of RNAP to move is directly confirmed by the structures inwhich jaws are opened or closed and by demonstration that the hingeddomain of RNAPII moves away from the RNA exit tunnel (Fu et al., 1999).Thus, it appears possible that the jaws, the active site channel, andthe RNA exit tunnel all may be able to open and closesemi-independently.

This leads to the obvious possibility that σ contacts to the flap, theβ′ coiled-coil, and β domain 3 hold open the active site channel and RNAexit tunnel (in a conformation similar to the Taq RNAP crystalstructure), that release of σ from these contacts allows these two partsof RNAP to close, and that this closure explains the change fromunstable initiating complexes to the stable TEC. In particular, acomplementary or induced fit between the inner surface of the activesite channel and the RNA:DNA hybrid is an appealing explanation for themajor energy of stabilization in the TEC.

Such a model explains several properties, of promoter complexes andTECs. In the σ-RNAP complex, the opening of the active site channelwould allow the entry of promoter DNA, possibly triggering closure ofthe jaws on the downstream DNA. Anchoring the DNA between RNAP'sdownstream contact and σ bound at the upstream face of RNAP couldfacilitate DNA strand separation by σ to form the transcription bubble.At this point, σ would continue to hold open the active site channel andRNA exit tunnel. When the RNA transcript grows to ˜8 nt, it will havefilled the rifampicin-binding site in β, its 5′ end will reach thetranscript separation point or rudder (see Zhang et al., 1999), and thegrowing RNA chain would encounter σ bound to β domain 3 and the β′coiled-coil. Further chain extension could require loss of thesecontacts and in the process allow the growing chain to pass under theflap as the RNA separates from the DNA template. Coincident orsubsequent loss of the σ-to-flap contact would then allow the flap toclose over the RNA exit tunnel, configuring RNAP for processive RNAsynthesis.

A requirement to release a in order to stably enclose the RNA:DNA hybridwithin the active site channel and the exiting RNA under the flap wouldexplain why σ and RNA compete for binding to RNAP-DNA complexes (Daubeand von Hippel, 1999) and why 8-9 nt RNAs are not stably in TECs when ahas been released (Chamberlin and Hsu, 1996; Korzheva et al., 1998 andreferences therein). Although the free energy of base pairing in theRNA:DNA hybrid (as well as the interaction of RNAP's jaws withdownstream DNA) probably makes a contribution to TEC stability (seeNudler, 1999 and references therein), these interactions alone do notappear sufficient since both already exist in initiating complexeswithout conferring TEC-like stability. An additional energeticcontribution made by locking a complementary protein surface tightlyaround the helical shape of the RNA:DNA hybrid best explains the TEC'sstability (Korzheva et al., 1998).

A terminator hairpin might destabilize the TEC by reopening the RNA exittunnel and active site channel of RNAP. In principle, wedging of theterminator hairpin under the flap domain could open the RNA exit tunnel;disruption of even 1 bp in the RNA:DNA hybrid could be sufficient toopen the active site channel if maintenance of its closed conformationdepends on a precise fit of the channel around the hybrid. In this view,termination would involve the reversal of structural transitionsobserved during initiation. This model is similar to that proposed forthe single-subunit T7 RNAP, where both TEC formation and terminationappear to involve movement of a loop in the N-terminal domain thatcontacts RNA (Lyakhov et al., 1997).

The idea that TEC destabilization at intrinsic terminators depends inpart on wedging open the flexible flap that covers the RNA exit runnelis consistent with the position of cross-linking to RNAP of a nascentRNA hairpin that is part of a transcriptional pause signal closelyrelated to intrinsic termination signals (Wang et al., 1997 andreferences therein). In TECs halted at this pause, the loop of the pausehairpin cross-links specifically to amino acid residues located at thetip of the flap domain (β domain 6) in the Taq RNAP structure (Wang etal., 1997). Thus, the pause hairpin-appears to form between the flap andthe body of the enzyme (partially within the RNA exit tunnel). Sopositioned, it could alter the active site channel's conformation,affect the position of the RNA:DNA hybrid, or both. This would explainhow the hairpin inhibits proper alignment of the of the 3′ OH with theactive site. At a terminator, the hairpin forms even closer to the RNA3′ end (7-9 nt away versus 11 nt for the pause; see Chan et al., 1997and references therein), and could wedge open the RNA exit tunnelsetting up release of the transcript.

The idea that termination requires opening of the active site channel issupported by the observations of TEC dissociation when a hairpin orantisense oligonucleotide pairs close enough to the RNA 3′ end to affecthybrid structure (position −8 or −9, see Artsimovitch and Landick, 1998;Korzheva et al., 1998; Yarnell and Roberts, 1999; Nudler, 1999 andreferences therein). This upstream edge of the hybrid may be directlyadjacent to contacts that lock the channel closed, so that a disruptionof base-pairing would open the channel. Thus, a terminator hairpin mightreturn RNA polymerase to a conformation similar to that of the promotercomplex, first by forming under the flap and opening the RNA exittunnel, and then by melting the −8 bp in the hybrid and disrupting theclosed conformation of the active site channel. Coupled with weak rU dAbase-pairing in the remaining hybrid and absence of σ (which maintainsthe transcription bubble in a promoter complex), these events could leadto rapid collapse of the bubble and TEC dissociation (Artsimovitch andLandick, 1998; Korzheva et al., 1998; Nudler, 1999 and referencestherein). Thus the new structure of RNAP is consistent with the ideathat termination could occur by an opening of RNAP followed bytranscription bubble collapse. However, an alternative model in whichhairpin formation pulls the RNA out of the enzyme without disruptingRNAP's structure remains equally viable (see Yarnell and Roberts, 1999).

The invention will be further described by the following non-limitingexamples.

EXAMPLE 1 Allosteric Control of RNA Polymerase

Materials and Methods

Crosslinking of the Pause Hairpin to RNA Polymerase

To confirm the assignment of βF906 as the crosslinking target of 5-iodoUin the pause hairpin loop, two types of experiments were performed.First, paused TECs containing either wild-type or βK909C mutant RNAPsand a wild-type pause hairpin loop derivatized with 5-iodoU and[α-³²P]CMP were prepared (FIG. 1A). The 5-iodoU crosslink was mapped bycleaving the crosslinked β subunit either to completion with trypsin orpartially with 2-nitro-5-thiobenzoic-acid (NTCB), and by separating thecleavage products on SDS polyacrylamide gels (8-16% gradient; FIG. 6A).Trypsin cleavage was performed prior to disruption of the TEC, whichresults in cleavage within β only at R903 and K909 (Boruhkov et al.,1991). As reported previously for wild-type (Wang et al., 1997),essentially all of the crosslinked RNA was attached to the fragmentβK909C RNAP from R903 to the C-terminus (trypsin cleavage at K909 isblocked by the substitution in this mutant). NTCB cleaves at Cysresidues, which are present at seven positions in wild-type β and eightin βK909C. Essentially all of the label was attached C-terminal of C838in both wild-type and mutant RNAPs, but was N-terminal of C909 in themutant β subunit as evidenced by the absence of radioactivity on theC909-to-C-terminus fragment and the appearance (relative to wild-type)of a radioactively labeled N-terminus-to-C909 fragment (FIG. 6A). Thus,the combined results described hereinbelow and the trypsin and NTCBcleavage results demonstrate unambiguously that 5-iodoU in the pausehairpin loop crosslinks to β904-908 (C-terminal of trypsin cleavage at903 and N-terminal of NTCB cleavage of 909; FIG. 6C). By the same logic,4-thioU placed at the same locations in the pause hairpin loop alsomapped between NTCB cleavages at C838 and C909 (FIG. 6B) and C-terminalof trypsin cleavage at R903. A minor fraction of the 4-thioUcrosslinking appears to be N-terminal of R903 (note FIG. 1B). This isnot surprising because 4-thioU crosslinks to a wider variety of aminoacid side chains than 5-iodoU. 4-thioU placed in the pause hairpin looplikely crosslinks to several amino-acids in the vicinity of F906, with aminor fraction being N-terminal of R903. Importantly, however, unlikethe 5-iodoU crosslink to F906, the 4-thioU crosslinks are retained inpaused TECs containing βF906A mutant RNAP (FIG. 6D). Some 4-thioUcrosslinking to β¢ also was detected (FIG. 6D), but the β¢ crosslink wasnot mapped (the part of β¢ likely to be nearest the hairpin loop is aZn²⁺, binding domain that is not resolved in the crystal structure(Zhang et al., 1999). Thus, the loss of crosslinking to β by 5-iodoU inthe βF906A mutant RNAP was not caused by repositioning of the flap-tiphelix relative to the pause hairpin loop because such a repositioningshould also have affected the 4-thioU crosslink. Rather, 5-iodoU mustfail to crosslink the F906A mutant RNAP because βF906 is the target of5-iodoU crosslinking.

Molecular Modeling

The coordinates of the tetraloop pause hairpin were assembled fromcoordinates of a canonical A-form RNA stem and a C-UUCG-G tetraloopdetermined by NMR by Colemenjaro and Tinoco (1999), which is essentiallyidentical to the C-UUCG-G tetraloop crystal structure recently reportedby Ennifar et al. (Ennifar et al., 2000). The hairpin was modeled into aTEC structural model (Korzheva et al., 2000, coordinates kindly suppliedby S. Darst) that was based on the crystal structure of Thermusaquaticus RNAP (Zhang et al., 1999). The hairpin was aligned with theTEC structure (see FIG. 2) by positioning the tetraloop uridines closeto fSF906 when the −11 nt remained in the location reported by Korzhevaet al. (2000). The locations of the NTP and second Mg²⁺ ion were modeledby overlaying the primer DNA strand, substrate dNTP, and catalytic Mg²⁺ions from the crystal structure of an active ternary complex of thelarge fragment of Thermus aquaticus DNA polymerase I (Li et al., 1998)with the RNA strand and single Mg²⁺ ion in the Thermus aquaticus RNAPstructure (Zhang et al., 1999).

Crosslinking of the −11 nt to RNA Polymerase

To map-1-nt crosslinks in RNA polymerase, the partial chemical cleavagestrategy of Grachev et al. (1989) was employed. Paused TECs wereprepared as described previously (Wang et al., 1997), except that4-thioU was incorporated at the −11 position rather than in the pausehairpin loop, using a mutant template that specified U at −11. A portionof the paused TECs was converted to rapidly elongating TECs byincubation with an antisense oligonucleotide that pairs to bases −33 to−15 relative to the pause RNA 3′ end. After UV irradiation at 365 nm toallow crosslinking, crosslinked β and β¢ subunits were separated in SDSpolyacrylamide gels (8-16% gradient), the fragments isolated, andpartial CNBr digestion performed. The partially cleaved products wereseparated on a second SDS polyacryalmide gel and compared to theproducts of control digestions of β or β¢ that had been radioactivelylabeled at their C- or N-termini, respectively, via heart-muscle-kinase(HMK)-sequence tags and reaction with HMK and [γ³²P]ATP (FIGS. 7 A andB). The rates of cleavage of the experimental samples were normalized tothe intrinsic rate of CNBr cleavage of β or β¢ by calculating the ratioof cleavage rates at corresponding positions in the experimental andcontrol samples (the average ratios for positions with significantradioactivity in the experimental samples were arbitrarily set to 1 toadjust for small differences in the overall rates of cleavage in theexperimental and control samples). Mapping protein crosslinking sites bynormalizing cleavage rates in partial chemical cleavage ladders wasfirst adopted by Korzheva et al. (2000). By correcting for the largedifferences in intrinsic cleavage rates at different amino acidresidues, it clearly reveals the drop in radioactivity intensity uponremoval of the crosslinked nucleic acid. In principal, it can revealmultiple crosslinks to a polypeptide as partial drops in cleavageintensity. In practice, apparent rates of cleavage at a given site inthe experimental and control samples can vary up to two-fold, possiblybecause the electrophoretic mobility of some polypeptides is shifted bythe crosslinked nucleic acid (FIGS. 7 A and B). Thus, reliableassignment of multiple crosslinks requires that relative band intensityin the experimental versus control samples drop for multiple, sequentialpositions. The position of the −11 nt was the same in the paused andnonpaused TECs. To locate it on the structure of RNAP, segments to whichthe crosslink was mapped were compared in the β and β¢ subunits(β1243-1273 and β¢238-298). The only overlap between these two segmentsin the crystal structure of RNAP (Zhang et al., 1999) occurs where aflexible portion of β¢ was unresolved in the structure (β¢249-260).Assuming maximal flexibility for this 12 amino-acid loop, it wasdetermined that the possible locations of the −11 nt that could accountfor crosslinking to both β1243-1273 and β¢238-298 were restricted to a6×9 Å area corresponding to β1250-1260. The −11 nt was also mapped byCys-specific partial cleavage (with NTCB) to β(838-1342) andβ¢(198-366). Paused and nonpaused TECs yielded identical cleavagepatterns, in agreement with the conclusion that the −11 nt does not movefrom the β1250-1260 region upon paused TEC formation.

Effect of Flap-Tip Helix on Pausing and NusA Regulation of Transcription

To assess the contribution of the flap-tip helix to pausing and to NusAregulation of transcription, halted TECs containing wild-type orβD(900-9009) RNAP were prepared from synthetic oligonucleotides usingthe procedure of Siderenkov et al. (1998) (FIG. 8A). Wild-type andβ-mutant TECs were assembled using complementary oligonucleotides(nontemplate strand:

(SEQ ID NO:1) CTATAGGATACTTACAGCCATCGAGAAACACCTGACTAGTCTTTCAGGCGATGTGTGCTGGAAGACATTCAGATCTTCC(Artismovitch et al., 2000). 100 nM template strand and RNA(UUUUUACAGCCAUC; SEQ ID NO:2) were annealed in 20 mM Tris-HCl, pH 7.9,20 mM NaCl, 0.1 mM EDTA, and then incubated with 100 nM RNAP in the samebuffer plus 5% glycerol, 5 mM MgC₁₂, 0.1 mM DTT, 50 μg BSA/ml for 10minutes at 22° C. After incubation at 37° C. with nontemplate DNA (250nM) for 10 minutes and then with 2.5 μM each ATP and GTP and 1 μM[a-₃₂P]CTP for 10 minutes to form C24 TECs, NTPs were adjusted to 10 μMGTP, 150 μM each ATP, UTP, and CTP, and pausing was assayed with orwithout NusA as described previously (FIG. 8B; Artsimovitch and Landick,2000). The flap-tip-helix deletion reduced the pause half-life two-foldin the absence of NusA (FIG. 8), but reduced hairpin stimulation ofpausing from 10-fold to 2-fold. NusA stimulated pause half-life about3-fold for the wild-type TECs, but had no effect on the βD(900-909)TECs. Further, NusA slowed arrival of wild-type TECs at the pause siteby 5-10 seconds by slowing nucleotide addition at the sites between C24and the pause, but had no effect on the time required for βD(900-909)TECs to transcribe from C24 to the pause. Thus, all NusA's effects ontranscription and pausing were eliminated by deletion of the flap-tiphelix. Other pause half-lives reported in FIG. 4 were determinedsimilarly or by forming initialed halted TECs on double-strandedtemplates containing a T7 A1 promoter (Artisomovitch et al., 1998) andthen assaying chain elongation as described herein.

Results

To test the rigid-body versus allosteric models, a pause hairpin wasstudied that inhibits nucleotide addition by a factor of 10 to 20 at theEscherichia coli his pause site. The his pause synchronizestranscription with translation in the attenuation control region of theE. coli his operon (Chan and Landick, 1993). The pause signal ismultipartite; interactions of downstream DNA, 3′-proximal RNA, and NTPsubstrate, together with the pause hairpin, inhibit nucleotide additionby a factor of about 100 (Chan and Landick, 1993). The pause hairpinloop substituted with 5-iodoU photocrosslinks strongly to RNApolymerase's β-subunit flap domain between residues 903 and 952, causingan unusual retardation of β during SDS-PAGE relative to RNA-β crosslinksin nonpaused TECs (Wang et al., 2000). This crosslink also occurs with a5-iodoU-substituted tetraloop pause hairpin (FIG. 1), which pausesequivalently to wild-type (Chan and Landick, 1993) and whose structureis known (Ennifar et al., 2000), or when 4-thioU is used in place of5-iodoU. 5-iodoU, but not 4-thioU, preferentially crosslinks to Phe,Tyr, Trp, Met, and His residues (Meisenheimer and Koch, 1967), which arefound between β903-952 only at F906 and F934 (FIG. 1B). RNA polymerasesubunits (α, N-terminally His₆-tagged wild-type or mutant β, and β′carrying a C-terminal intein and chitin-binding domain) wereco-overexpressed in E. coli. After sonication and capture of RNApolymerase on a chitin matrix (New England Biolabs), RNA polymerase wasrecovered by DTT-mediated intein cleavage. βF934A RNA polymerase pausedequivalently to wild-type RNAP; βF906A RNAP pausing was more sensitiveto competition by Cl⁻; βΔ(900-909) RNAP pausing was reduced at both lowand high [Cl⁻] (FIG. 4). TEC synthesis and photocrosslinking wereperformed as described previously (Wang et al., 1997).

F906 is the likely target of hairpin-loop crosslinking because thestrong, shifted-β band disappeared when a βF906A, but not a βF934A,mutant RNAP was used (FIG. 1C). To confirm this, it was established that(i) 5-iodoU or 4-thioU in the hairpin loop crosslinked betweentrypsin-cleavage at 903 and 2-nitro-5-thiobenzoic-acid-cleavage at 909(in βK909C RNA polymerase) and (ii) 4-thio-U crosslinking was retainedin the βF906A mutant, showing that F906A does not alter hairpin-flapinteraction.

F906 is located in a short c-helix at the tip of RNA polymerase's flapdomain (Zhang et al., 1999). The flap is flexibly connected to RNApolymerase, held open by lattice forces in a crystal structure, andthought to close over the exiting RNA in a TEC (Zhang et al., 199;Korzheva et al., 2000; Finn et al., 2000), probably by contact of ahydrophobic patch on the flap-tip helix to the main body of RNApolymerase (FIG. 2). When the pause hairpin was modeled into a TECstructure (Korzheva et al., 2000), using the reported position of the−11 nt (the nt immediately 3′ of the hairpin) and the loop contact tothe flap-tip helix, the hairpin fit under the open flap domain (FIG.2B). Thus, unless the flap closes in the TEC, the steric clash predictedby the rigid-body model would not occur. If the exit channel closes asexpected, the hairpin could pull the RNA (including the −11 nt) throughthe exit channel and away from the active site, while retaining loopcontact to the flap-tip helix. Alternatively, the hairpin could open theexit channel, potentially generating an allosteric signal.

To distinguish these possibilities, three predictions of the rigid-bodymodel were tested: (i) hairpin formation should move the −11 nt; (ii)lengthening the 3-nt spacer between the hairpin and hybrid should reducethe hairpin's ability to pull RNA away from the active site and thus tostimulate pausing; and (iii) stabilizing the hairpin by lengthening itsstem should increase its stimulation of pausing.

To map its contacts to RNA polymerase, the −11 nt was substituted with4-thioU (5-iodoU crosslinked poorly at −11). Separately, the 3′-terminalnt was substituted with 5-iodoU. Photocrosslinking was performed in thepaused TEC, or after hybridization of an antisense oligonucleotide thatconverts it to a rapidly elongating TEC (FIG. 3A; Artsimovitch andLandick, 1998). As predicted by both models, 3′-nt contacts changed uponpaused TEC formation (β′:β ratio changed, FIG. 3A), but no change wasdetected in the −11 nt position even when it was mapped to a 6 Å by 9 Åarea where the mapped segments of β and β′ crosslinking overlapped(FIGS. 3B-E). This also is the previously reported −11-nt location(Korzheva et al., 2000), validating this modeling of the pause hairpin.Importantly, were hairpin formation to move −11 more than one nt,crosslinking to this small area would not be maintained (nt insingle-stranded, extended RNA are separated by ≧7.5 Å; FIG. 3C), andeven slight movement likely would alter the β′:β ratio (see FIG. 3A).

To test effects of spacer length, spacer length was varied from 2 nt to5 nt (FIG. 4A). When the spacer length was increased to 4 nt,hairpin-dependent pausing increased, directly contradicting therigid-body model. Hairpins with 2-nt or 5-nt spacing supported pausingif NusA was present.

To test lengthening the hairpin stem, the stem was changed from 5 to 8bp (using the optimal 4-nt spacer). Hairpin-dependent pausing was lostwithout NusA, again contradicting the rigid-body model (FIG. 4A).Although hairpin shape and location clearly are important for pausing,all the spacer and stem results are readily explained by specifichairpin interactions with RNA polymerase and NusA.

Together, these results and previous findings make a convincing caseagainst the rigid-body model: (i) the pause hairpin cannot be imitatedby antisense oligonucleotide-pairing to nascent-RNA (Artsimovitch andLandick, 1998); (ii) unraveling of the RNA:DNA hybrid, required ifhairpin formation pulls RNA through the exit channel, does not occur ina paused TEC (Lee and Landick, 1992); (iii) the hairpin creates stericclash with RNA polymerase, as required by the rigid-body model, only ifthe flap closes in the TEC (FIG. 2), yet the hairpin fails to move the−11 nt as it must if the exit channel remains closed (FIG. 3); and (iv)increasing spacer length increases pausing (FIG. 4A), whereas increasinghairpin stem-length reduces pausing (Chan et al., 1997, FIG. 4), bothopposite to the rigid-body model's predictions. Thus, the steric effectsof a pause hairpin on RNA translocation through a rigid RNA polymerasealone cannot explain hairpin-dependent pausing. The hairpin must causesome conformational change in RNA polymerase, for which flap or clampmovement is the apparent trigger.

Because NusA enhancement of pausing requires the pause hairpin (Chan andLandick, 1993; Artsimovitch and Landick, 2000), made suboptimal pausesignals work better (FIG. 4A), and is modulated by the structure of thehairpin loop (Chan and Landick, 1993; e.g., compare tetraloop to wildtype in FIG. 4A). NusA also may act via interaction with the flap-tiphelix, either directly or by contacting the hairpin loop. When NusA wasadded to paused TECs containing 5-iodoU-substituted hairpin loops,strong, retarded-gel-mobility crosslinking to βF906 was replaced byweaker crosslinking to NusA (lanes 2 and 3, FIG. 4B). Both the NusA andshifted-β crosslinks disappeared when the hairpin was disrupted with anantisense oligonucleotide, and did not occur in nonpaused TECs at −9(lanes 4-7, FIG. 4B).

To test whether the flap-tip helix was required for pausing and NusAaction, a RNA polymerase lacking the helix (βΔ(900-909)) was preparedand tested. In the absence of NusA, the helix deletion reduced hairpinstimulation of pausing from about 10-fold to about 2-fold (FIG. 4A). Thehelix deletion completely abolished NusA-enhancement of pausing (FIG.4A), and NusA's ability to slow transcription elsewhere. Thus, theflap-tip helix is required for regulation of pausing by NusA and formost of the pause hairpin's effect.

Therefore, NusA likely stabilizes pause hairpin-flap interaction, which,by opening the RNA exit channel, may allosterically affect RNApolymerase's active site. Interaction of the composite NusA-β-subunitsurface with RNA may stabilize RNA structures and explain NusA's abilityto accelerate cotranscriptional folding of RNA (Pan et al., 1999).

How might an allosteric signal generated by flap contact affectcatalysis in the active site, which is 65 Å from the flap-tip helix? Theflap domain connects to RNA polymerase through a two-stranded,antiparallel beta-sheet (the connector). The connector runs along theactive-site cleft to highly conserved amino acids in the active site(E813, D814, and K1065; FIG. 3). E813 and D814 may chelate the Mg²⁺ ionbound to the substrate NTP; K1065 contacts the α phosphate of the3′-terminal RNA nt; substitution of E813 or K1065 disrupts catalysis(Mustaev et al., 1991; Sagitov et al., 1993). Therefore, the pausehairpin may affect catalysis by moving the flap and, by way of theconnector, critical residues in RNA polymerase's active site.Alternatively, hairpin formation could open the active site cleft bymoving the clamp domain. Conversely, flap or clamp movement and possiblyhairpin formation could be inhibited when NTPs bind efficiently (becausebound NTP would constrain the position of E813/D814), and may be coupledto movements of parts of RNA polymerase that form the active-site cleftand downstream DNA jaws (FIG. 2; Mooney and Landick, 1999).

Definition of the flap-tip helix as an allosteric site on RNA polymeraseprovides a new framework for understanding RNAP's regulation. σ alsobinds RNA polymerase's flap (Gruber, T. I. Artsimovitch, K Geszvain, R.Landick and C. Gross, unpublished data); a may open the RNA exit channelto thread RNA into the channel: σ release may allow the channel to closefor efficient transcript elongation (Mooney and Landick, 1999). Likepause hairpins, terminator hairpins probably also open the RNA exitchannel, rather than pull RNA out of RNA polymerase, and then dissociatethe TEC by invading the RNA:DNA hybrid, opening the active-site cleft,and triggering collapse of the transcription bubble (Korzheva et al.,2000; Artsimovitch and Landick, 2000). Finally, eukaryotic RNApolymerases also contain a flap domain, making the flap an attractivetarget for both prokaryotic and eukaryotic regulators of transcription.

EXAMPLE 2 Flap Domain Interactions

FIG. 9 summarizes the transcription cycle. σ factors and NusA alternateinteraction with RNAPs in a cycle of binding that allows RNAP torecognize promoters when bound by σ factors and to synthesize RNAmolecules properly when bound by NusA. As discussed above, the presenceof the flap domain in RNAP is important for RNAP to support bacterialgrowth, and the flap-tip helix of the flap domain helps accessorytranscription factors bind to RNAP and carry out its function. Theflap-tip helix is important for the function of both the major σinitiation factor (σ⁷⁰ in Escherichia coli) and the essential elongationfactor NusA.

Effect of Substitutions on Flap-Tip Helix Interactions

Amino acid substitutions in the flap-tip helix (Example 1) can preventbacterial growth. To determine whether bacterial growth is dependent onthe hydrophobicity of the flap-tip helix or a particular amino acidsequence, constructs were prepared with various substitutions in theflap-tip domain. The substitutions were in a hydrophobic patch on theflap-tip helix (FIG. 10).

A simple assay was employed to test the effect of flap-tip helixsubstitutions on bacterial growth. Cells were transfected with aplasmid, such as pRL702 (FIG. 11), which includes a RNAP β subunitcontaining the substitution. pRL702 encodes the β subunit under thecontrol of a trc promoter, which in turn is regulated by the lacrepressor expressed from the same plasmid (Amann et al., 1988). WhenIPTG is added to growth medium, expression of the substituted β subunitis turned on and the resultant β subunit outcompetes the chromosomallyencoded, wild-type β subunit for assembly into RNAP. If the RNAP withthe substituted β subunit cannot support transcription, no furtherbacterial growth occurs. For example, serial dilutions of a culturegrown without IPTG are plated onto rich medium agar plates containingIPTG. The magnitude of the growth defect is reflected in the reductionin the number of bacterial colonies that form on the IPTG-containingplate.

Table 1 shows the relative plating efficiencies of bacteria carryingplasmids that encode β subunits with substitutions in the flap-tiphelix. The data show that the specific amino acids present in thehydrophobic patch are not important to bacterial growth. Rather, thenonpolar nature of the patch is important. For instance, substitution ofleucine 901, a central residue in the hydrophobic patch, with alaninehas a much lesser effect than substitution of leucine 901 with polaramino acids (lysine, arginine or glutamine). A deletion of the flap-tiphelix (Δ(900-909)) is as lethal as the polar substitutions at 901,showing that these substitutions likely disrupt some essential activityof the flap-tip helix, rather than result in a toxic effect. Mostsubstitutions at positions 902, 905, and 906 are less toxic than similarsubstitutions at 901, but the overall pattern of these effects indicatesthat the hydrophobic patch makes a hydrophobic interaction with someother molecule, e.g., part of RNAP, that is quite significant for properfunction of the enzyme.

TABLE 1 Plating efficiency* of bacteria with plasmids that encodesubstitutions in the flap-tip helix. Substitution Plating EfficiencyNone 0.7 Δ(900-909) 0.0001 LA901 1.5 (slow) LK901 0.00015 LQ901 0.00017LR901 0.00016 LA902 0.7 LP902 0.2 (slow) LQ902 0.7 IA905 2.1 (slow)IK905 0.00019 IN905 1.25 (slow)  IS905 3.2 (slow) FA906 0.6 (slow)*Plating efficiency is the number of colonies formed on IPTG-containingmedium divided by number of colonies formed on medium lacking IPTG.“(slow)” substitutions are ones that significantly inhibit bacterialgrowth, causing formation of small colonies.

Initiation and the Flap-Tip Helix

To understand which aspect of RNAP function was compromised by theflap-tip substitutions, the properties of the lethal deletion mutant,βΔ(900-909), were studied. Synthesis of the α, β, and β′ subunits ofRNAP were controlled by a T7 RNAP promoter on a plasmid that encodes allthree subunits (derivatives of pIA312; FIG. 11). The is β′ subunitcarried a self-cleaving protein segment, intein, fused to its C-terminusfollowed by a chitin-binding domain (CBD). When expression of thesubunits was induced in cells that express T7 RNAP under IPTG control,the mutant RNAPs assembled in the cells and can be purified from celllysates by passing the lysates over a chitin resin which binds themutant RNAPs and then cleaving the intein portion of β′ by the additionof DTT. To eliminate the small amount of RNAP that contained wild-type βsubunit encoded by the chromosome from these preparations, twoapproaches were used. One approach took advantage of a hexahistidine tagthat was present at the N-terminus of the mutant β subunit to affinitypurify the mutant RNAP on Ni²⁺-agarose. Another approach took advantageof a S. aureus protein A tag that had been engineered into thechromosomal copy of rpoB (the gene for the β subunit; Opalka et al.,2000)), to remove the RNAP with the chromosomally encoded β subunit byadsorbsion onto human IgG-agarose (Tarromina et al., 1996).

Purified RNAPs were then used for in vitro transcription reactions. TheβΔ(900-909) mutant RNAP was found to be competent for elongation oftranscripts and for termination of transcription (Toulokhonov et al.,2001). For instance, βΔ(900-909) mutant RNAP was as active on a poly(dAdT) template as wild-type RNAP (assayed as described in Gross et al.,1976), but the mutant was incapable of initiation from a strong E. coliRNAP promoter, the T7 A1 promoter (FIG. 12A).

TABLE 2 Relative activity of wild-type and mutant RNAPs on poly d(AT).poly(dA · dT) activity* RNAP −σ70 +σ70 wt 1.0 ± 0.05 2.0 ± 0.1 Δ(900-909) 0.95 ± 0.02  0.9 ± 0.05 LR901 1.2 ± 0.05 1.5 ± 0.07 LQ901 1.2± 0.05 1.4 ± 0.07 LP902 1.2 ± 0.05 1.5 ± 0.09 IN905 1.0 ± 0.04 1.3 ±0.02 FA906 1.0 ± 0.14 2.1 ± 0.15 *cpm of [³H]UMP incorporated into acidinsoluble RNA using poly(dA · dT) as a template. Values for mutant RNAPsare divided by those for a comparable amount of wild-type RNAP.

The βΔ(900-909) mutant RNAP displayed a defect in the stimulation ofpoly(dA dT) by σ⁷⁰, which ordinarily increases poly(dA dT) by a factorof 2-3. Fisher and Blumenthal (1980) showed that trypsin cleavage of theβ subunit was protected by σ⁷⁰. This trypsin cleavage site wassubsequently mapped to R903 and K909 (Borukhov et al., 1991), residuesin or directly adjacent to the flap-tip helix. Thus, the defect intranscription initiation by βΔ(900-909) RNAP on the T7 A1 promoter mayreflect some defect in the way RNAP interacts with σ factors.

To dissect the basis of the transcriptional defect of the flap-tip helixmutants, transcription from a T7 A1 promoter DNA template was comparedto transcription from a similar template that contained a so-calledconsensus galP1 promoter, which does not require interaction of σ⁷⁰ withthe −35 promoter element (Kumar et al., 1994; Severinova et al., 1998).These transcription templates were produced from the plasmid pIA171(Artsimovitch et al., 2000) by PCR with oligonucleotides that yielded,either the wild-type T7 A1 promoter of a galP1 consensus −10 derivativewith the sequence shown in FIG. 12D. Transcription reactions wereconducted by mixing 25 nM RNAP with 50 nM DNA template in 1×transcription buffer (Artsimovitch et al., 2000), incubating the mixturefor 10 minutes at 37° C., and then for an additional 10 minutes afterthe addition of ApU dinucleotide (to 50 μM), ATP, [³²P]CTP, and GTP (to10 μM each), and heparin (to 100 μg/ml). The radiolabeled RNA productswere separated by electrophoresis through a denaturing polyacrylamidegel (8 M urea, 19:1 acrylamide:bisacrylamide, 44 mM Tris-borate, pH 8.3,2.5 mM Na₂EDTA). The amount of radioactive CMP incorporated into the 29nt transcripts that form on these templates in the absence of UTP wasmeasured using an Molecular Dynamics Phosphorimager and the amounts ofproduct produced from the mutant RNAPs relative to the wild-type RNAPwas calculated from these values (FIGS. 12A and C). Based on this data,it was concluded that the flap-tip helix is important for initiation onpromoters that require interaction of a factor with a −35 promotersequence, such as the T7 A1 promoter, but not from promoters such as thegalP1 consensus −10 promoter that do not require interaction of σ factorwith a −35 promoter element. The magnitude of the defect is greatestwhen then the flap-tip helix is deleted (Δ(900-909), or when theflap-tip helix hydrophobic patch is most completely disrupted byreplacement of L901 with arginine.

These results strongly support the hypothesis that the flap-tip helixmust interact with region 4 of σ factors to facilitate transcriptionalinitiation at promoters that require σ factor contact to the −35promoter element.

Flap-Tie Helix Contacts with Region 4.2 of σ⁷⁰

To test directly whether the flap-tip helix contacts region 4 of σfactors, the ability of trypsin to cleave the flap-tip in RNAPs eitherin the absence of σ factors or in the presence or wild-type a or alacking region 1 or region 4 was tested (FIG. 14). RNAPs were incubatedwith trypsin in the absence (FIGS. 14 A and C) or presence (FIGS. 14 Band D) of a 2-fold molar excess σ⁷⁰. Samples were removed at the timesindicated, mixed with trypsin inhibitor, and then separated byelectrophoresis through native polyacrylamide gels (4-15% Phast gels,Amersham-Pharmacia). After staining the gels with silver, the amounts ofβ(1-903) and β(1-909) polypeptide produced were quantified using a CCDcamera image and the extent of cleavage was calculated as the fractionof full-length β subunit cleaved at the times sampled. From these data(Table 3) an apparent cleavage rate could be estimated by non-linearregression.

To test for the effect of the flap-tip helix hydrophobic patch on theability of σ⁷⁰ to protect the β subunit against trypsin cleavage, therates of trypsin cleavage of wild-type RNAP to an RNAP bearing the LQ901substitution were compared (Table 3). The LQ901 substitution was chosenbecause position 901 exhibited the strongest effects of flap-tipsubstitutions and because the L to Q change does not create a trypsincleavage site (trypsin cleaves after lysine and arginine residues). Fromthe results, it was concluded that the flap-tip hydrophobic patch isimportant in allowing σ factor interaction in a way that protects the βsubunit against trypsin cleavage.

To test which region of or σ⁷⁰ interacts with the flap-tip helix, theeffect of removing parts of σ⁷⁰ on its ability to protect the flap-tipagainst trypsin cleavage was examined. Removal of region 4 of σ, theportion known to interact with the −35 promoter sequence (Siegele etal., 1989; Gordella et al., 1989; Pombroski et al., 1992), caused acomplete loss of σ's ability to protect against trypsin cleavage,whereas removal of region 1 of σ caused a slight increase in the rate ofcleavage (Table 3).

TABLE 3 Rates of β subunit cleavage as a function of flap-tip helixsequence or σ subunit. β cleavage rate − σ⁷⁰ β cleavage rate + σ⁷⁰ RNAPσ s⁻¹ × 10³ L901Q wt 12 ± 2* 10 ± 2   Wild-type wt 9.2 ± 0.1 1.6 ± 0.2 ″ Δ1 — 6.2 ± 0.4  ″ Δ4 — 11 ± 0.4 ″ Δ1, Δ4 — 11 ± 0.5Flap-Tip Helix Contacts with NusA

RNAP with a deletion of the flap-tip helix (Δ(900-909)) is completelyresistant to the action of NusA protein (Toulokhonov et al., 2001).Further, part of NusA is positioned near the flap-tip in pausedtranscription complexes (Toulokhonov et al., 2001), making it likelythat NusA interacts with the flap-tip helix. Using a trypsin cleavageassay, it was found that NusA protects the flap-tip helix from trypsincleavage (FIG. 15), similar to data for σ⁷⁰. Further, the L901Qsubstitution in the flap-tip hydrophobic patch lessens the ability ofNusA to protect the flap-tip helix from trypsin cleavage. Thus, likeσ⁷⁰, NusA also interacts with the flap-tip, likely replacing the σcontacts made during initiation with NusA contacts made duringelongation. The NusA-flap-tip helix interaction likely differs somewhatfrom the σ⁷⁰-flap-tip helix interaction, because the L901Q substitutionhas a bigger effect of the σ interaction than on the NusA interaction.

CONCLUSION

From the results described hereinabove, both σ⁷⁰ and NusA make contactsto the flap-tip helix that are important to their functions. At leastfor σ⁷⁰, this interaction requires principally a hydrophobic patch inthe flap-tip helix, rather than a particular amino acid sequence at theflap-tip. Since the interactions of both σ⁷⁰ and NusA with the flap-tipare important for bacterial viability, the flap-tip helix is anexcellent target for isolation or design of antibacterial compounds.Compounds that bind the flap-tip helix should block the function oftranscription factors essential for bacterial viability and thus killbacteria. Moreover, the flap-tip helix is a small target for design ofinhibitors and is highly conserved among bacteria. Therefore, it shouldbe relatively easy to obtain antibiotics from any compound that binds tothe flap-tip helix, antibiotics which are likely effective against awide variety of bacteria. Because the flap-tip helix is required forinteractions with multiple transcription factors (e.g., σ initiationfactors and NusA), it should be relatively difficult for bacteria toacquire natural resistance to antibiotics targeted to the flap-tip helixby mutations that alter the amino acid sequence of the flap-tip helix.Such mutations would also compromise interactions with the essentialtranscription factors and thus block their function.

EXAMPLE 3 Methods To Identity Inhibitors of Flap-Tip Helix Interactions

There are three likely interacting partners of the flap-tip helix ofEscherichia coli RNA polymerase: the σ⁷⁰ initiation factor, the NusAtranscription elongation factor, and nascent RNA secondary structures.As described above, both σ⁷⁰ and NusA are unable to function when theflap-tip helix is deleted. Therefore, inhibitors of these interactionsblock transcription initiation in E. coli and, because the flap-tiphelix is conserved among bacteria, but not eukaryotes, it is likely thatsuch inhibitors function as broad-spectrum antibiotics with specificityfor bacterial and not human or animal cells. It also is likely thatother transcription factors, such as alternative a factors also requirethe flap-tip helix for function. The exemplary assays described belowcan be used to detect interactions of transcription factors with theflap-tip helix and to screen for inhibitors of these interactions thatwould serve as lead compounds for antibiotic design. The same assays canbe adapted to characterize interactions of transcription factors otherthan σ⁷⁰ and NusA and to look for specific inhibitors of thoseinteractions.

There are three classes of assays that can be performed. In the first,the flap-tip helix is left intact in RNA polymerase and its interactionsare monitored by one of several methods described below. In the second,the flap-tip helix or the entire flap domain is displayed separatelyfrom RNA polymerase and the interactions of proteins like σ⁷⁰ or NusAare assayed by methods that detect simple binding. In this second case,because only the flap domain or flap-tip helix is presented as a bindingtarget, it is not necessary to distinguish interactions of the flap-tiphelix from interactions with other parts of RNA polymerase. In the thirdclass of assay, the flap-tip helix or the entire flap domain isdisplayed separately from RNA polymerase and used to look directly formolecules that bind the flap-tip helix. Because bulky molecules thatbind the flap-tip helix are likely to block σ⁷⁰ or NusA function, suchmolecules are good candidates for lead compounds in antibiotic design.

I. Alterations of RNA polymerase. NusA, and σ⁷⁰ to FacilitateInteraction Assays

The assays can take advantage existing or easily constructed mutants inthe flap-tip helix, σ⁷⁰, and NusA as controls to distinguishinteractions of the flap-tip helix from interactions with other parts ofRNA polymerase. Mutants in the flap-tip helix were constructed using aplasmid, pRL702, that expresses the β subunit of RNA polymerase carryinga hexahistidine tag and hemagglutinin epitope at its N-terminus. Thisplasmid is similar to pRW308 (Weilbaecher et al., 1994), except thatpRW308 expresses a wild-type version of the β′ subunit of E. coli RNApolymerase. A deletion of the flap-tip helix, β(Δ900-909), and pointmutants LR901, LQ901, LP902, and IQ905, that are unable to function withσ⁷⁰, were prepared. The deletion β(Δ900-909) is unable to function withNusA. As described below, LQ901 directly blocks interaction of σ⁷⁰ withthe flap-tip helix. These mutants of the flap-tip helix, either inintact RNA polymerase, or in polypeptides expressing just the β subunit,the flap domain, or the flap-tip helix serves as powerful controls toidentify molecules that interact specifically with the flap-tip helix.

In addition to its interaction with the flap-tip helix, σ⁷⁰ is known tobind tightly to a coiled-coil domain in the β′ subunit of RNA polymerasethrough so-called region 2 of σ⁷⁰ (Arthur et al., 2000; Burgess et al.,1998; Arthur and Burgess, 1998). This is the strongest interaction ofσ⁷⁰ with RNA polymerase. This interaction can be prevented frominterfering with a flap-tip helix binding assay, either by deleting ormutating region 2 of σ⁷⁰ or by deleting or mutating the coiled-coil inβ′.

Likewise, NusA is known to interact with the C-terminal domain of the αsubunit of RNA polymerase through NusA's C-terminal domain (Mah et al.,2000). This is the strongest interaction of NusA with RNA polymerase,but actually is dispensable for its activity in vivo if NusA is producedin cells at higher concentration (Schauer et al., 1987). Thisinteraction of NusA is prevented from interfering with a flap-tip helixbinding assay either by deleting or mutating the C-terminal domain ofNusA or the C-terminal domain of the α subunit of RNA polymerase.

Desired mutants in RNA polymerase, σ⁷⁰, or NusA can be prepared usingthe standard methodology of oligonucleotide-directed mutagenesis ofplasmid DNA (Sugimoto et al., 1989).

II. Class 1 Assays

A. Trypsin Cleavage Protection

One of the simplest ways to assay for interaction of a macromoleculewith the flap-tip helix is protection against trypsin cleavage. At lowconcentration, trypsin cleaves RNA polymerase in only three locations,two of which, R903 and K909, are in or near the flap-tip helix Borukhovet al., 1991). Cleavage of R903 or K909 is readily detected byseparation of RNA polymerase subunits on an SDS-polyacrylamide gel,whereupon trypsin cleavage of R903 or K909 generates two new bandscorresponding to the larger N-terminal portion of the β subunit and thesmaller C-terminal portion of the β subunit. Addition of either σ⁷⁰ orNusA protects the flap-tip helix against trypsin cleavage (Fisher etal., 1980; unpublished observations). Further this is specific to thebiologically relevant interaction of the flap-tip helix because σ⁷⁰ nolonger protects the flap-tip helix of a mutant RNA polymerase (L901Q)that is compromised for σ⁷⁰ function and σ⁷⁰ interaction with theflap-tip helix (unpublished observations).

This assay is mostly useful to confirm suspected interactions orinhibitors of interaction. Large molecules suspected of interacting withthe flap-tip helix may afford protection against trypsin cleavage andcould by this inhibition of trypsin cleavage. Smaller molecules thatinhibit interaction of σ⁷⁰ or NusA with the flap-tip helix could preventσ⁷⁰ or NusA from inhibiting trypsin cleavage of the flap-tip helix.Therefore small molecule inhibitors of σ⁷⁰ or NusA interactions with theflap-tip helix could be detected by the loss of σ⁷⁰ or NusA protectionof the flap-tip helix against trypsin cleavage.

B. Fluorescence Resonance Energy Transfer (FRET)

Interactions with the flap-tip helix could be assayed by FRET if thedonor or acceptor fluorophore were located near the flap-tip helix andthe other partner were located on σ or NusA. When interaction occursfluorescence energy would be transferred between the two fluorophoresowning to their proximity. If the interaction were lost, the FRET signalwould decrease. This assay would be adaptable to mass screening forinhibitors of interactions of the flap-tip helix with σ or NusA(Matsumoto et al., 2000). Any compound that inhibited such aninteraction would decrease FRET signal and this decrease could bedetected in fluorescence instruments that are designed for massscreening. Many methods exist for specific attachment of fluorophoresand these can be adapted to locate fluorophores on σ⁷⁰ or NusA (see, forexample, Callaci et al., 1999). Specific attachment of fluorophores orother probes to the flap-tip helix is described below.

C. Fluorescence Perturbation Assay

Interaction of σ⁷⁰ or NusA with the flap-tip helix also could perturbthe fluorescence signal from a fluorophore attached to or near theflap-tip helix, even if the σ⁷⁰ or NusA did not itself contain afluorophore. The interaction itself could either increase or decreasethe fluorescence of a fluorophore attached to the flap-tip helix bychanging the microenvironment around the fluorophore. This assay alsowould be amenable to mass screening. Any compound that interfered withinteraction of σ⁷⁰ or NusA with the flap-tip helix could eliminate theincrease or decrease in fluorescence caused by this interaction and bedetected in spectrofluorometers designed for high-throughput assays.

D. Crosslinking Assays

Crosslinking moieties attached to the flap-tip helix can be used todetect molecules that interact with the flap-tip helix by transfer ofradioactive or chemical tags to the interacting molecule (see, e.g.,Chen et al., 1994 for an example of this method). Thus, molecules thatinhibit flap-tip helix interactions could be detected by interferencewith crosslinking between a suitable reagent attached to the flap-tiphelix and a target protein.

E. Tethered Protease or Nuclease Assays

Chemical or enzymatic proteases or nucleases that are attached to theflap-tip helix could be used to detect molecules that interact with theflap-tip helix by cleavage or degradation of the interacting molecules.An example of this methodology is described in Owens et al. (1998).Thus, molecules that inhibit flap-tip helix interactions could bedetected by interference with cleavage or degradation of a targetprotein by a suitable reagent attached to the flap-tip helix.

F. Attachment of Chemical Moieties to the Flap-Tip Helix (“derivatives”)

Some of the assays described above depend on specific attachment ofchemical moieties to the flap-tip helix. A general method for thisattachment is as follows. A derivative of the β subunit of E. coli RNApolymerase was prepared that contains a cysteine residue in place of thenormal lysine residue at position 909 adjacent to the flap-tip helix.These mutant β subunits are expressed from the plasmid pRL702.Additional cysteine substitutions in the β subunit can be prepared usingpRL702 by oligonucleotide-directed mutagenesis. The cysteine residue(s)can be reacted with a large variety of commercially availablefluorophore or other chemical compounds that react with the freesulfhydryl group (see, for example, Callaci et al., 1998 and Owens etal., 1998). To conduct the attachment reaction under conditions wherethe desired cysteine is the only one available for reaction, RNApolymerase is reconstituted from individual subunits and split subunits,one of which a fragment of the β subunit on which the desired cysteineresidue is the only cysteine residue. Functional RNAP can bereconstituted from α, β′, β(1-643), and β(643-1342) subunits or subunitfragments (Severinov et al., 1995). β(643-1342) contains the entire flapdomain, but only three Cys residues (at position 764, 770, and 838).These residues were changed to serine, isoleucine, and serine,respectively, in intact β subunit and were found to support growth of E.coli. Therefore, sulfhydryl-reactive compounds can be reacted with aβ(643-1342) fragment in which the tlineonly cysteine residue is presentin or near the flap-tip helix, such as the cysteine at 909. Then thesederivatized β subunit fragments are used to reconstitute RNA polymerasein vitro for use in the assays described above.

III. Class 2 Assays

A. Localization Assays

In localization assays, the two interacting partner proteins are assayedwith one protein partner immobilized on a solid support and the secondpartner protein tagged in some way to reveal its interaction with theimmobilized protein. There are a large number of variations tolocalization assays, all of which are applicable to assayinginteractions of the flap-tip helix. Either the flap-tip helix containingprotein or the interacting protein (here NusA or σ⁷⁰) can serve as theimmobilized protein or the marked protein that interacts from solution.In the simplest implementation of these assays, either the flap domain(consisting of E. coli β subunit residues from about 830 to about 1058),of the flap tip helix alone (consisting of at least E. coli β subunitresidues 897-907) is attached to the solid support. For instance, theflap-tip helix protein could be attached to the wells of a multiple wellplastic plate, either by direct absorption, chemical reaction withderivatized plate wells, binding to another molecule (such as anantibody) attached to the plate wells, or derivatization of the flap-tiphelix protein so that it can be attached specifically ornon-specifically to the plate well. Binding of the σ⁷⁰ or NusA proteinto the immobilized flap-tip helix protein can be detected in many way,some of which are (i) using a radioactively, fluorescently,luminescently, or phosphorescently labeled σ⁷⁰ or NusA protein or a σ⁷⁰or NusA-protein derivative and detecting binding by the presence ofradioactivity, fluorescence, luminescence, or phosphorescence in theplate welts after they are exposed to the σ⁷⁰ or NusA protein and thenwashed; (ii) using a radioactively, fluorescently, luminescently, orphosphorescently labeled antibody to σ⁷⁰ or NusA protein or a σ⁷⁰- orNusA-protein derivative and detecting binding by the presence ofradioactivity, fluorescence, luminescence, or phosphorescence in theplate wells after they are exposed to the σ⁷⁰ or NusA protein, washed,then exposed to the antibody, and then washed again; (iii) using afluorescently, luminescendy, or phosphorescently labeled σ⁷⁰ or NusAprotein or a σ⁷⁰- or NusA-protein derivative and a fluorescently,luminescently, or phosphorescently labeled σ⁷⁰ or NusA protein or a σ⁷⁰-or NusA-protein derivative, and then detecting binding by the transferof energy between one of the molecules that is excited with specificwavelength electromagnetic radiation and the second, which is notexcited but whose emissions are monitored (a fluorescence resonanceenergy transfer, FRET, or luminescence resonance energy transfer, LRET,assay); (iv) using a change in fluorescence polarization when afluorescently labeled protein interacts with the second protein that isderivatized in such a way as to make it significantly larger than thefluorescently labeled protein (a fluorescence anisotropy assay; see,e.g., Owicki, 2000); and (v) attaching one of the proteins to a solidsupport containing a scintillant, and then detecting the binding of theother protein, which is radioactively labeled, by activation of thescintillant by the proximity of the radioactive emissions created by thebinding reaction (a scintillation proximity assay, SPA, see Cook et al.,1992).

Using localization assays such as those described in this section orvariants of these assays, it is possible to screen for compounds thatdisrupt interactions of σ⁷⁰ or NusA with the flap-tip helix by addingcompounds or mixtures of compounds to the bound proteins or during thebinding reaction, and then identifying those compounds that preventgeneration of the signal that indicates binding. These assays areadaptable to high-throughput screening and could be used to screen largelibraries of candidate inhibitor compounds.

B. In Vivo Assays of Protein Interactions

A second method to identify interactions of the flap-tip helix with σ⁷⁰or NusA is to detect the interactions in vivo by a biological signalgenerated upon interaction of the two proteins. A well-characterizedexample is the yeast two-hybrid assay (Fields and Sternglanz, 1994). Inthis case σ⁷⁰ or NusA protein or a σ⁷⁰- or NusA-protein derivative isattached to either a DNA-binding domain or an activation domain and theflap-tip helix protein attached to the other domain. If interactionoccurs, recruitment of the activation domain to the DNA binding domain,which is localized near a promoter in living cells, activatestranscription of a reporter gene, which in turn generates a visiblesignal, such as changing the color of colonies of the organism growingon a suitable solid support. This assay also is adaptable tohigh-throughput screening. Cells that generate the positive signal uponinteraction of σ⁷⁰ or NusA with the flap-tip helix protein are placed inthe wells of a multiple well plastic plate together with candidateinhibitors of the interaction or mixtures of inhibitors, and then thepresence or absence of interaction is detected by automated platereaders.

An in vivo assay based on a −35 promoter sequence can also be used. Forexample, one cell expresses a construct with a −35 promoter sequencelinked to one marker gene and another cell expresses a construct with a−10 promoter sequence linked to the same marker gene. Alternatively, onecell expresses a construct with a −35 promoter sequence linked to onemarker gene and a construct with a −10 promoter sequence linked to adifferent marker gene. Agents which inhibit the expression from the −35promoter construct in cells expressing wild-type β, e.g., inhibition ata level similar to that from the −35 promoter construct in cellsexpressing β with a mutation in the flap-tip helix in the absence of theagent, and not from the −10 promoter construct are candidate inhibitorsof the flap-tip helix. In vitro transcription reactions which measurethe amount or level of the transcription product from the −35 or −10promoter sequences may also be employed.

IV. Class 3 Assays (Direct Detection of Molecules that Bind the Flap-TipHelix)

In this class of assay, compounds that bind the flap-tip helix areidentified directly and then subsequently tested for their ability tointerfere with NusA or σ⁷⁰ function using in vitro transcription assays.In these assays the derivatives of the flap-tip helix that fail to bindσ⁷⁰ or NusA, such as the β(Δ900-909) deletion can be used as a counterscreen to eliminate compounds that do not bind specifically to theflap-tip helix.

A. Localization Assays

The same methods described above for localization assays can be used tolook for molecules that interact with the flap-tip helix by attachingthe candidate compounds to a solid support and then using a derivativeof the flap-tip helix that can be detected upon binding. Alternatively,the flap-tip helix can be attached to a solid support and theinteracting compounds can be detected provided that candidate compoundsare derivatized in a manner that allows their detection upon binding tothe solid support.

B. Yeast Two-Hybrid Assay

The flap-tip helix can be attached to either the DNA binding domain ofthe activator domain in a yeast-two-hybrid assay σ⁷⁰ or NusA protein ora σ⁷⁰- or NusA-protein derivative (Fields and Sternglanz, 1994).Libraries of compounds attached to the other partner in the yeasttwo-hybrid assay can then be screened to identify interacting compounds.

C. Phage or Cell Display Assay

In the phage- or cell-display assay (Sidney, 2000 andWesterlund-Wikstrom, 2000), the flap-tip helix protein is attached to asolid support and then libraries of compounds attached either to theoutside of phage particles and genetically encoded in the correspondingphage genome, or attached to the outside of bacterial or yeast cells andgenetically encoded in the corresponding bacterial or yeast genomes, areallowed to interact with the immobilized flap-tip helix protein. Aftersuitable washing, the bound phage, bacteria, or yeast cells arerecovered by gentle elution and the genetic information encoding theinteracting molecule is recovered from them by conventional methods.These assays are particularly useful because the phage or cellsrecovered from one round of selection or counterselection can beamplified by growth in suitable conditions so that they can be subjectedto multiple rounds of selection. It also is possible increase thestingency of the binding steps, or decrease the stringency of thecounterselection steps, or to introduce a mutagenetic step in thesegrowth steps so that the multiple rounds of selection andcounterselection can serve to select out the phage or cells that encodemolecules that bind the flap-tip helix bind with optimal properties.Proteins in which the flap-tip helix is deleted can serve as a usefulcounterscreen in these assays, for instance by exposing the selectedphage or cells that are eluted from the first round of binding to animmobilized protein in which the flap-tip helix is mutated or deleted.Using this counterselection approach, phage or cells that stick to thiscontrol matrix could be discarded prior to proceeding to the next roundof amplification and selection because they bind nonspecifically.

D. In Vitro Selection and Directed Evolution Assays

Potential lead compounds for creation of transcription inhibitors thattarget the flap-tip helix also could be created by in vitro selection ofnucleic acids (RNA or DNA or other nucleic acids or nucleic-acidderivatives that can be replicated in vitro or in vivo) by repeatedrounds of binding and replication of nucleic acids that adhere to animmobilized flap-tip helix protein (Szostak, 1997; Joyce, 1997). Thismethod, sometimes referred to as SELEX (Gold et al., 1997) can generatenovel compounds (sometimes referred to as aptamers; Brody and Gold,2000) that inhibit RNA polymerase function by tightly binding theflap-tip helix. The counterselection strategy described above also couldbe used with SELEX strategies to discard nonspecifically bindingcompounds. These compounds can be used directly or can be used fordesign of similar molecules with improved properties. The aptamers couldbe particularly useful because they could be expressed inside bacterialcells when encoded by DNA or RNA such that activation of expression ofthe compounds inhibit growth of the bacteria or kill the bacteria.

E. mRNA Display Assay

Potential lead compounds for creation of transcription inhibitors thattarget the flap-tip helix also could be created by mRNA displaytechnologies (Cho et al., 2000; Wilson et al., 2001). These methodsallow generation of libraries of peptide aptamers of much greatercomplexity than is possible by phage display because the peptide aptameris attached in vitro directly to the mRNA that encodes it, so thataptamers recovered after binding to an immobilized flap-tip helixprotein carry with them the genetic information that can be replicatedand used to synthesize additional aptamers. As with SELEX, theseaptamers could potentially be subjected to multiple rounds of selection,replication, and even mutagenesis to evolve improved binding properties.The counterselection strategy described in section V.C also could beused with mRNA display strategies to discard nonspecifically bindingcompounds. Peptide aptamers discovered by this method could be used fordesign of similar molecules with improved properties. They also could beexpressed inside bacterial cells when encoded by DNA or RNA such thatactivation of expression of the peptide aptamer or its derivatives wouldinhibit growth of the bacteria or kill the bacteria.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1. A method to identify one or more agents which inhibit or prevent thebinding of a moiety to the flap-tip helix of the β subunit of core RNApolymerase, comprising: a) providing a complex formed by contacting theone or more agents with isolated core RNA polymerase, or an isolated βsubunit of RNA polymerase or a portion thereof which comprises theflap-tip helix; b) contacting the complex with a moiety whichspecifically binds the flap-tip helix, wherein the moiety is a nucleicacid aptamer, a peptide aptamer or an initiation factor; and c)detecting or determining whether the one or more agents inhibit orprevent the binding of the moiety to the flap-tip helix.
 2. A method toidentify one or more agents which inhibit or prevent the binding of amoiety to the flap-tip helix of the β subunit of core RNA polymerase,comprising: a) providing a complex formed by contacting the one or moreagents with a moiety which specifically binds the flap-tip helix,wherein the moiety is a nucleic acid aptamer, a peptide aptamer or aninitiation factor; b) contacting the complex with isolated core RNApolymerase, or an isolated β subunit of RNA polymerase or a portionthereof which comprises the flap-tip helix; and c) detecting ordetermining whether the one or more agents inhibit or prevent thebinding of the moiety to the flap-tip helix.
 3. A method to identify oneor more moieties which bind to the flap-tip helix, comprising: a)contacting a peptide comprising the flap-tip helix or a portion thereofwith the one or more moieties; and b) detecting or determining whetherthe one or more moieties specifically bind to the flap-tip helix.
 4. Themethod of claim 1, 2 or 3 wherein the moiety is a nucleic acid aptamer.5. The method of claim 1, 2 or 3 wherein the moiety is a peptideaptamer.
 6. The method of claim 1, 2 or 3 wherein the moiety is aprokaryotic protein.
 7. The method of claim 3 wherein the moiety is atranscription factor.
 8. The method of claim 7 wherein the transcriptionfactor is an initiation factor.
 9. The method of claim 7 wherein thetranscription factor is an elongation factor.
 10. The method of claim 1,2 or 3 wherein moiety is σ.
 11. The method of claim 10 wherein theinitiation factor is σ⁷⁰.
 12. (canceled)
 13. The method of claim 1, 2 or3 wherein the portion includes residues corresponding to residues 900 to909 of the β subunit of E. coli RNA polymerase.
 14. The method of claim1, 2 or 3 wherein the portion includes residues corresponding toresidues 897 to 907 of the β subunit of E. coli RNA polymerase.
 15. Themethod of claim 1, 2 or 3 wherein the portion includes residuescorresponding to residues 830 to 1058 of the β subunit of E. coli RNApolymerase.
 16. The method of claim 1 or 2 wherein core RNA polymerase,the β subunit or a portion thereof is labeled or is capable of beingbound by a detectable label.
 17. The method of claim 16 wherein afluorophore is attached to or near the flap-tip helix.
 18. The method ofclaim 16 wherein core RNA polymerase, the β subunit or portion thereofis capable of being bound by a labeled antibody.
 19. The method of claim1, 2 or 3 wherein the moiety is labeled or is capable of being bound bya detectable label.
 20. The method of claim 19 wherein the moiety iscapable of being bound by a labeled antibody.
 21. The method of claim 3wherein the peptide is labeled or is capable of being bound by adetectable label.
 22. The method of claim 1 or 2 wherein fluorescenceresonance energy transfer is employed to detect or determine whether theagent inhibits or prevents binding.
 23. The method of claim 1 or 2wherein luminescence resonance energy transfer is employed to detect ordetermine whether the agent inhibits or prevents binding.
 24. The methodof claim 1 or 2 wherein trypsin cleavage and SDS-PAGE are employed todetect or determine whether the agent inhibits or prevents binding. 25.The method of claim 1 wherein core RNA polymerase or the β subunit orportion thereof is attached to a solid substrate.
 26. The method ofclaim 25 wherein the solid support comprises a scintillant and themoiety is radioactively labeled.
 27. The method of claim 2 wherein themoiety is attached to a solid substrate.
 28. The method of claim 27wherein the solid support comprises a scintillant and core RNApolymerase, β subunit or portion thereof is radioactively labeled. 29.The method of claim 3 wherein the peptide is attached to a solidsupport.
 30. The method of claim 29 wherein the solid support comprisesa scintillant and the moiety is radioactively labeled.
 31. The method ofclaim 3 wherein the one or more moieties are products encoded by arecombinant phage, virus, or cell.
 32. The method of claim 1, 2 or 3wherein a protease or nuclease is attached to or near the flap-tiphelix.
 33. The method of claim 1, 2 or 3 wherein a cross-linking moietyis attached to or near the flap-tip helix.
 34. An agent identified bythe method of claim 1 or
 2. 35. A method to inhibit the growth of aprokaryotic cell, comprising: contacting the cell with an effectiveamount of the agent of claim
 34. 36. An isolated and purified portion ofthe β subunit of RNA polymerase which binds to NusA or σ.
 37. Theisolated and purified portion of the β subunit of claim 36 whichcomprises a portion selected from the group consisting of residuescorresponding to residues 830 to 1058, residues 897 to 907, and residues900 to 909 of the β subunit of E. coli RNA polymerase.
 38. A method toidentify one or more agents that which inhibit or prevent the binding ofa prokaryotic protein to the flap-tip helix of the β subunit of core RNApolymerase, comprising: a) contacting the one or more agents with arecombinant cell which expresses a first fusion polypeptide and a secondfusion polypeptide, wherein the first fusion polypeptide comprises theflap-tip helix and a first polypeptide and the second fusion polypeptidecomprises the prokaryotic protein or a fragment thereof whichspecifically binds the flap-tip helix and a second polypeptide, whereinthe binding of the first polypeptide to the second polypeptide yields adetectable signal; and b) detecting or determining whether the one ormore agents inhibits or prevents the signal.
 39. The method of claim 38wherein either the first or the second polypeptide comprises a DNAbinding domain.
 40. The method of claim 38 wherein either the first orthe second polypeptide comprises an activation domain.
 41. A moietyidentified by the method of claim
 3. 42. A method to identify one ormore agents which inhibit or prevent transcription from a promoter thatis dependent on a −35 sequence for activity, comprising: a) contactingthe one or more agents with a composition for transcription comprisingwild-type RNA polymerase and a first construct comprising a firstpromoter that is dependent on a −35 sequence to promote transcriptionand operably linked to an open reading frame for a gene; and b)identifying an agent that inhibits or prevents transcription from thefirst construct relative to transcription by wild-type RNA polymerasefrom a second construct comprising a second promoter that promotestranscription independent of the presence of a −35 sequence, whichsecond promoter is operably linked to an open reading frame for a gene,thereby identifying an agent that inhibits or prevents transcriptionfrom a promoter that is dependent on a −35 sequence for activity. 43.The method of claim 42 wherein the one or more agents are contacted witha recombinant cell augmented with the first construct.
 44. The method ofclaim 42 wherein the level of transcription from the first construct inthe presence of the agent is substantially the same as the level oftranscription by a mutant RNA polymerase comprising a mutant flap-tiphelix from a third construct comprising a promoter that is dependent ona −35 sequence to promote transcription and operably linked to an openreading frame for a gene.
 45. The method of claim 42 wherein the agentdoes not inhibit transcription by a mutant RNA polymerase comprising amutant flap-tip helix from a third construct comprising a promoter thatpromotes transcription independent of the presence of a −35 sequence andoperably linked to an open reading frame for a gene.
 46. The method ofclaim 42 wherein the open reading frame in the first construct is anopen reading frame for a marker gene or a selectable gene.
 47. Themethod of claim 42 wherein the open reading frame in the secondconstruct is an open reading frame for a marker gene or a selectablegene.
 48. The method of claim 42 wherein the open reading frame in thefirst construct is different that the open reading frame in the secondconstruct.
 49. The method of claim 42 wherein the recombinant cellfurther comprises the second construct and wherein the open readingframe in the first construct is different that the open reading frame inthe second construct